Methods of testing for intracellular pathogens

ABSTRACT

A first intracellular pathogen in a biological sample that may contain more than one intracellular pathogen is studied by a method comprising the steps of (i) contacting the sample with a population of cells in the presence of an agent inhibiting the reproduction of a second intracellular pathogen; (ii) incubating the cells under conditions that permit the reproduction of the first intracellular pathogen; and (iii) testing material arising from step (ii) for the first intracellular pathogen.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.13/583,619, with an international filing date of Mar. 7, 2011; which isa National Phase of International Patent Application No.PCT/IB2011/001057, filed Mar. 7,2011; which claims the benefit of U.S.Provisional Patent Application No. 61/339,764, filed Mar. 8, 2010; thedisclosures of which are herein incorporated by reference in theirentirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name:223002125901SeqListing.txt, date recorded: Sep. 22, 2016, size: 2 KB).

TECHNICAL FIELD

This invention relates to methods for studying intracellular pathogensin biological samples. More particularly, the invention relates tomethods of studying biological samples containing more than oneintracellular pathogen, and methods of observing or testing for thepresence of a first intracellular pathogen in the presence of a secondintracellular pathogen.

BACKGROUND ART

It may be of interest to study two different intracellular pathogens inthe same biological sample, for example a sample isolated from a patientor from a cell culture, or to observe one intracellular pathogen in thepresence of other intracellular pathogens. However, often the presenceof one such pathogen interferes with observations of other pathogens.

For example, when the effects of intracellular pathogens are studied inassays leading to general phenotypic results (“phenotypic assays”), theobservables are not necessarily specific for a particular pathogen. Forexample in virology, a plaque observed in a cell culture cannotnecessarily be attributed to a given virus if a further pathogen capableof causing such plaques is also present.

However, it is often desirable to employ general phenotypic assays.Other, more specific methods of observing pathogens suffer fromdifferent drawbacks. Often, more specific methods are not asstraightforward or rapid to perform. Such other methods may also requirespecific reagents which impose further limitations.

Immunological assays, for example, require antibodies to be available.The utility of immunological approaches may further be limited whenantibodies are cross-reactive for multiple pathogens that may be presentin the sample. For some diagnostic purposes, broad cross-reactivity ofantibodies to related pathogens may be desirable. On the other hand,such cross-reactivity may make it impossible to study related andco-existing pathogens independently by immunological methods.

PCR-based testing for pathogens can be more specific. However, PCR-basedmethods require genomic sequences to be known for each pathogen ofinterest. Primers must be designed and optimized for each pathogen ofinterest.

The requirement for specific antibodies or primers limits theflexibility of both immunological and PCR-based methods, and inparticular their utility for observing pathogens when the nature of thepathogen is not known. The utility of these specific methods maymoreover be limited in the case of rapidly evolving pathogens, e.g.,certain viruses, because mutations may occur in epitopes recognized byantibodies or in the sequences recognized by PCR primers.

It is an object of the invention to provide further and improved methodsfor studying intracellular pathogens of interest in a biological sampleindependently of other intracellular pathogens.

DISCLOSURE OF THE INVENTION

The invention provides a process for testing for a first intracellularpathogen in a biological sample, comprising the steps of:

-   -   (i) contacting the sample with a population of cells in the        presence of an inhibitory agent, wherein said agent inhibits the        reproduction of a second intracellular pathogen;    -   (ii) incubating the cells tinder conditions that permit the        reproduction of said first intracellular pathogen; and    -   (iii) testing material arising from step (ii) for said first        intracellular pathogen.

According to the methods of the invention, biological samples can betested rapidly. The invention allows biological samples to be tested forintracellular pathogens in a simple, straightforward manner, e.g., by ageneral phenotypic assay. Preferred methods allow an intracellularpathogen of interest to be studied independently of the (actual orsuspected) presence in the sample of a further intracellular pathogenthat would lead to the same, equivalent or similar result in suchassays.

The invention also allows biological samples to be tested forintracellular pathogens in circumstances

-   -   wherein the presence in the sample of one or more intracellular        pathogens (e.g., the first intracellular pathogen of the methods        defined above and in the claims) is unknown, and/or    -   wherein the genomic sequences of one or more intracellular        pathogens in the sample (e.g., the first intracellular pathogen        of the methods defined above and in the claims) are unknown,        and/or    -   wherein antibodies to one or more intracellular pathogens in the        sample (e.g., the first or the second intracellular pathogens of        the methods defined above and in the claims) are unavailable,        and/or    -   an antibody to the second intracellular pathogen is not        employed,    -   wherein antibodies to two or more intracellular pathogens in the        sample are cross reactive, and/or    -   it is desirable to obtain test results rapidly.

The processes of the invention encompass embodiments wherein a testresult of step (iii) is obtained within 12 months, or, e.g., within 9months, 8 months, 7 months, 6 months, 5 months, 4 months, 3 months, 2months, 1 month, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4days, 3 days, 2 days, 1 day, or sooner.

Intracellular Pathogens of the Methods of the Invention

The first and second intracellular pathogens of the methods of theinvention may encompass one or more intracellular pathogen selected froma prokaryote a bacterium/bacteria), a eukaryote (including aprotozoon/protozoa or a fungus/fungi), and/or a virus/viruses. The firstintracellular pathogen is different from the second intracellularpathogen. The first and second pathogens may differ in any aspect thatallows them to be differentially inhibited. For example, they may differin order, family, genus, species, sub-species and/or strain.

The first intracellular pathogen may be a prokaryote, a eukaryote and/ora virus. Preferably, the first intracellular pathogen is a virus.

The second intracellular pathogen may be a prokaryote, a eukaryoteand/or a virus. Preferably, the second intracellular pathogen is avirus.

The invention encompasses various types of assays, for example, in oneembodiment, the first intracellular pathogen comprises a prokaryote(e.g., one or more of the preferred prokaryotes listed below), aeukaryote (e.g., one or more of the preferred eukaryotes listed below)and/or a virus (e.g., one or more of the preferred viruses listedbelow), and the second intracellular pathogen comprises a prokaryote(e.g., one or more of the preferred prokaryotes listed below).

In a further embodiment, the first intracellular pathogen comprises aprokaryote (e.g., one or more of the prokaryotes fisted below), aeukaryote (e.g., one or more of the eukaryotes listed below) and/or avirus (e.g., one or more of the viruses listed below), and the secondintracellular pathogen comprises a eukaryote (e.g., one or more of thepreferred eukaryotes listed below).

In a further embodiment, the firm intracellular pathogen comprises aprokaryote (e.g., one or more of the prokaryotes listed below), aeukaryote (e.g., one or more of the eukaryotes listed below) and/or avirus (e.g., one or more of the preferred viruses listed below, e.g., aparainfluenzavirus and/or e.g., an influenzavirus, a rhinovirus, arotavirus, an enterovirus, a human immunodeficiency virus, a simianimmunodeficiency virus, and/or a norovirus), and the secondintracellular pathogen comprises a virus (e.g., one or more of thepreferred viruses listed below, e.g., un influenza virus, and/or e.g. arhinovirus, a rotavirus, an enterovirus, a human immunodeficiency virusor simian immunodeficiency virus, and/or a norovirus, and/or e.g. aparainfluenzavirus).

According to the invention, prokaryotes may include intracellularpathogens selected from Bartonella, Bordetella, Brucella Chlamydiales,Chlamidiacae, Chlamydiae, Chlamydophila, Ehrlichia, Escherichia,Francisella, Legionella, Listeria, Mycobacterium, Mycobacterium,Rickettsiae, Salmonella, Shigella, Streptococcus, Yersinia.

In particular, prokaryotes may include intracellular pathogens selectedfrom Bartonella grahamii, Bordetella pertussis, Brucella spp., Chlamydiatrachomatis, Chlamydophila pneumoniae, Chlamydophila pecorum,Chlamydophila psittaci, Chlamydophila abortus, Chlamydophila felis,Chlamydophila caviae, Ehrlichia chaffeenis, Escherichia coli, Fracisellatularensis, Legionella pneumophila, Listeria monocytogenes,Mycobacterium tuberculosis, Mycobacterium leprae, Rickettsiae,Salmonella enterica Serovar Typhimurium, Salmonella typhi, Shigellaflexneri, Shigella dysenteriae, Streptococcus pyogenes, Yersinia pestis.

According to the invention, eukaryotes may include intracellularpathogens selected from Babesia, Cryptococcus, Cryptosparidium, Eimeria,Histoplasma, Leishmania, Plasmodium, Theileria, Toxoplasma, Trypanosama.

In particular, eukaryotes may include Babesia spp., Cryptococcusneoformans, Cryptosporidium parvum, Eimeria spp., Histoplasmacapsulatum, Leishmania mexicana, Leishmania donovani, Plasmodiumberghei, Plasmodium yoelii, Theileria spp., Toxoplasma gondii,Trypanosoma cruzi.

According to the invention, viruses may include

-   -   Pneumovirinae, such as the Pneumovirus genus, including        respiratory syncytial virus (RSV);    -   Morbilliviruses of the Paramyxoviridae family, such as the        measles virus;    -   Enteroviruses of the Picornaviridae family, such as the        Coxsackie viruses, echoviruses and enteroviruses;    -   mammalian Reoviridae, in particular orthoreoviruses (e.g.        mammalian reoviruses) and/or rotaviruses;    -   Avian Reoviridae, in particular orthoreoviruses, such as avian        reoviruses;    -   Metopneumoviruses of the Paramyxoviridae family, such as human        metapneumovirus (HMPV);    -   Rubulaviruses of the Paramyxoviridae family, such as mumps        virus;    -   Togaviridae such as Rubellavirus;    -   Coronaviridae, human coronaviruses, such as SARS coronavirus;    -   Rhinoviruses of the Picornaviridae family;    -   M-strains of Rhinovirus;    -   Varicella Zoster virus (VZV), also known as human herpes virus 2        (HHV3);    -   Polymaviridae, such as the SV-40 polyomavirus, the BK        polyomavirus JC polyomavirus,    -   porcine circoviruses;    -   porcine picornaviruses;    -   swine vesicular disease virus (SVDV);    -   Teschen-Talfan virus;    -   Parvoviruses, such as canine parvovirus (CPV), or porcine        parvoviruses, or Bocaviruses, e.g., human bocavirus;    -   Orthomyxoviridae, in particular influenza virus, e.g., influenza        A virus, influenza B virus and influenza C virus.    -   Parainfluenza viruses (PIV), Paramyxoviridae paramyxovirinae,        Parainfluenzavirus type 1, Parainfluenzavirus type 2,        Parainfluenzavirus type 3, Parainfluenzavirus type 4,        Parainfluenzavirus type 5;    -   Herpesviridae, herpes simplex virus 1 and 2;    -   Adenoviridae, such as the adenoviruses, including human and        simian adenovirus,    -   avian circoviruses;    -   an immunodeficiency virus such as human immunodeficiency virus        (HIV) or simian immunodeficiency virus (SIV);    -   noroviruses; and/or    -   Birnaviridae, such as infectious bursal disease virus (also        known as gumboro virus).

Thus, the first and/or second said first intracellular pathogen maycomprise one or more intracellular pathogens selected from Bartonella,Bordetella, Brucella, Chlamydia, Ehrlichia, Escherichia, Francisella,Legione Listeria, Mycobacterium, Mycobacterium, Rickettsiae, Salmonella,Shigella, Streptococcus, Yersinia, Babesia, Crypococcus,Cryptosporidium, Eimeria, Histoplasma, Leishmania, Plasmodium,Theileria, Toxoplasma, Tyrpanosoma, Pneumovirinae, the Pneumovirusgenus, respiratory syncytial virus (RSV), Morbilliviruses of theParapnyroviridae family, the measles virus, Enteroviruses of thePicornaviridae family, Coxsackie viruses, echoviruses and enteroviruses,mammalian Reoviridae, avian Reoviridae, orthoreoviruses, rotaviruses.Metapneumoviruses of the Paramyxoviridae family, human metapneumovirus(HMPV). Rubulaviruses of the Paramyxoviridae family, mumps virus,Togaviridae, Rubellavirus, Coronaviridae, human coronaviruses, SARScoronavirus, Rhinovineses of the Picornaviridae family, M-strains ofRhinovirus, Varicella Zoster virus (VZV), human herpes virus 2 (HHV3),Polyomaviridae, SV-40 polyomavirus, the BK polyomavirus JC polyomavirus,porcine circoviruses, porcine picornaviruses, swine vesicular diseasevirus (SVDV), Teschen-Talfan virus, Parvoviruses, canine parvovirus(CPV), porcine parvoviruses, Orthomyxoviridae, in particular influenzavirus, influenza A virus, influenza B virus and influenza C virus,Parainfluenza viruses (PIV), Paramyxoviridae paramyxovirinae,Parainfluenzavirus type 1, Parainfluenzavirus type 2, Parainfluenzavirustype 3, Parainfluenzavirus type 4, Parainfluenzavirus type 5,Herpesviridae, herpes simplex virus 1 and 2, Adenoviridae, adenoviruses,human and simian adenovirus, avian circoviruses, human immunodeficiencyvirus, simian immunodeficiency virus, norovirus, birnaviridae,infectious bursal disease virus (also known as gumboro virus).

For example, the second intracellular pathogen may preferably be, orcomprise, a coronavirus, e.g., a SARS coronavirus, an enterovirus, arotavirus, a rhinovirus, a human immunodeficiency virus, a simianimmunodeficiency virus, or a norovirus. In further preferredembodiments, the second intracellular pathogen may be, for instance, aninfluenza virus, e.g., an influenza A virus or an influenza B virus.

The method is not limited to any particular type or strain of virus. Forexample, if the second intracellular pathogen is an influenza virus or astrain thereof, the virus may be influenza A virus, influenza B virusand influenza C virus. Preferred influenza A strains in the context ofthe invention include strains of subtypes H1N1 (e.g., human and/or swineH1N1), H1N2 (e.g., human and/or swine H1N2), H2N2, H2N3, H3N1, H3N2(e.g., human and/or swine H3N2), H5N1, H7N2, H7N3, H7N7, H9N2, H10N7).Viruses in the methods of the invention may also be pandemic strains(i.e. strains to which the vaccine recipients and the general humanpopulation are immunologically naive), such as H1 (e.g., H1N1), H2, H5,H7 or H9 subtype strains (in particular of influenza A virus). Thus, inthe methods of the invention, an influenza A virus may have HA subtypeH1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16.The invention may be used with an influenza A virus having NA subtypeN1, N2, N3, N4, N5, N6, N7, N8 or N9.

The virus, e.g. an influenza A virus, may include one or more RNAsegments from a A/PR/8/34 virus (typically 6 segments from A/PR/8/34,with the HA and N segments being from a vaccine strain, i.e. a 6:2reassortant). It may also include one or more RNA segments from aA/W/SN33 virus, or from any other virus strain useful for generatingreassortant viruses for vaccine preparation. Typically, the virus may bea strain that is capable of human-to-human transmission, and so thestrain's genome will usually include at least one RNA segment thatoriginated in a mammalian (e.g. in a human or swine) influenza virus. Itmay include NS segment that originated in an avian, human or swineinfluenza virus.

The virus may be attenuated. The virus may be temperature-sensitive. Thevirus may be cold-adapted. The virus may be a reassortant strain, andmay have been obtained by reverse genetics techniques [e.g. 1-5].

The methods of the invention may be used to test for the presence orabsence of the first intracellular pathogen. That is, it may be unknownwhether the first intracellular pathogen is present in the biologicalsample to be tested. The first intracellular pathogen may be present inthe biological sample. The first intracellular pathogen may equally wellnot be present in the biological sample.

The second intracellular pathogen may or may not be present in thebiological sample. Preferably, the second intracellular pathogen isknown to be present in the biological sample. Alternatively, it may beunknown whether the second intracellular pathogen is present in thebiological sample to be tested.

In preferred embodiments, the second intracellular pathogen is a virus,and it is unknown whether the first intracellular pathogen is present inthe biological sample, i.e., the method of the invention is used to testfor the presence or absence of the first intracellular pathogen in thepresence of a virus.

The biological sample of the methods of the invention may also comprise,or be tested for, a third, fourth, fifth or further intracellularpathogen. Any such third or further intracellular pathogen may be asdescribed above fur the first or second intracellular pathogens. Inpreferred embodiments, it is unknown whether a third, fourth, fifth orfurther intracellular pathogen is present in the biological sample. Inpreferred embodiments, the methods of the invention are used to test forthe presence or absence of a multitude of intracellular pathogens, e.g.,for a third, fourth, fifth or further intracellular pathogen.

The Biological Sample

The invention can be used with various different biological samples. Thesample may contain, e.g., as the first and/or second intracellularpathogen, a virus of interest. Such a sample may be tested for thepossible presence of a first intracellular pathogen (e.g., a firstvirus) while the sample also contains a second intracellular pathogen(e.g., a second virus).

Laboratory Samples.

Clinical samples taken from patients e.g. from patients presentingsymptoms of an infection, from whom respiratory swabs are taken to checkfor possible new pathogens, e.g., new viral strains. The pathogen may,e.g., be any of the viruses listed above, for example an enterovirus, acoronavirus, a herpesvirus or an influenza virus.

Viral seeds, including master seed and working seeds. Such seed lots areused and tested during biologics production, where they form the basisof viral growth. The virus may be any of the viruses listed above, forexample an enterovirus, a coronavirus, a herpesvirus or an influenzavirus.

The presence of the second intracellular pathogen (e.g., a virus) maythus be desired, while the presence of the first intracellular pathogenis not desired. The method of the invention may be a test to confirm theabsence of the first intracellular pathogen.

Likewise, the presence the second pathogen (e.g., virus) may be known,white the presence of the first intracellular pathogen is not desiredand/or known.

Preferably, the sample is used solely for screening or testing purposes,rather than for testing within the process of manufacturing from saidsample. The methods may advantageously be combined with other knowntesting methods.

The biological sample may also be, or may be derived from, anembryonated egg, e.g., an embryonated chicken egg, including theallantoic cavity, or a chick embryo.

The biological sample may also be, or be obtained or derived from, acell or tissue culture, e.g. a growing cell or tissue culture, or anon-growing cell or tissue culture. Such a cultures may be a cell lineof mammalian origin. Suitable cells of mammalian origin include, but arenot limited to, hamster, cattle, primate (including humans and monkeys)and dog cells. Various cell types may be used, such as kidney cells,fibroblasts, retinal cells, liver cells, tuna, cells, etc. Examples ofsuitable hamster cells are the cell lines having the names BHK21 orHKCC. Suitable monkey cells are e.g. African green monkey cells, such askidney cells as in the Vero cell line. Suitable dog cells are e.g.kidney cells, as in the MDCK cell line. Thus suitable cell/linesinclude, but are not limited to: MDCK; CHO; 293T; BHK; Vero; MRC-5;PER.C6; WI-38; etc.

Preferably, said biological sample comprises or is derived frommammalian cells.

Preferred mammalian cell lines include: MDCK cells [6-9], derived fromMadin Darby canine kidney; Vero cells [10-12], derived from Africangreen monkey (Cercopithecus aethiops) kidney; or PER.C6 cells [13],derived from human embryonic retinoblasts. These cell lines are widelyavailable e.g. from the American Type Cell Culture (ATCC) collection[14], from the Coriell Cell Repositories [15], or from the EuropeanCollection of Cell Cultures (ECACC). For example, the ATCC suppliesvarious different Vero cells under catalog numbers CCL-81, CCL-81.2,CRL-1586 and CRL-1587, and it supplies MDCK cells under catalog numberCCL-34. PER.C6 is available from the ECACC under deposit number96022940.

The original MDCK cell line is available from the ATCC as CCL-34, butderivatives of this cell line may also be used. For instance, reference6 discloses a MOCK cell line that was adapted for growth in suspensionculture (‘MDCK 33016’, deposited as DSM ACC 2219). Similarly, reference16 discloses a MDCK-derived cell line that grows in suspension inserum-free culture (B-702′, deposited as FERM BP-7449), Reference 17discloses non-tumorigenic MDCK cells, including ‘MDCK-S’ (ATCCPTA-6500), ‘MDCK-SF101’ (ATCC PTA-6501), ‘MDCK-SF102’ (ATCC PTA-6502)and ‘MDCK-SF103’ (PTA-6503), Reference 18 discloses MDCK cell lines withhigh susceptibility to infection, including ‘MDCK.5F1’ cells (ATCCCRL-12042). Any of these MDCK cell lines can be used.

The sample be also be, or be obtainable from avian cell lines [e.g.refs, 19-21], including avian embryonic stem cells [19,22] and celllines derived from ducks (e.g. duck retina), or from Hens. Suitableavian embryonic stem cells, include the EBx cell line derived fromchicken embryonic stem cells, EB45, EB14, and EB14-074 [23], Chickenembryo fibroblasts (CEF), can also be used, etc.

The Population of Cells

In the method of the invention, a biological sample to be tested iscontacted with a population of cells, in vivo or in vitro. Thepopulation of cells may be, or may be comprised in, an organism,including an embryo, e.g., a mammal, a rodent, mouse, rat, guinea pig,hamster, rabbit, chick, or non-human primate. The population of cellsmay also be cells in culture. Preferably, the population of cells is acell culture (cultured cells). The population of cells may also be, forexample, an embryonated egg, e.g.., an embryonated chicken egg,including the allantoic cavity, or a chick embryo.

Generally, the population of cells may be as described above for thebiological sample. Accordingly, the population of cells, or culturedcells, may be a cell line of mammalian origin. Preferably, saidpopulation of cells comprises or is derived from mammalian cells. Inpreferred embodiments of the invention, both the biological sample andthe population of cells comprise, or are derived from, mammalian cells.

Suitable cells of mammalian origin are as described above for thebiological sample. These include MDCK cells, Vero cells or PER,C6 cells.Any of the cell lines described above in connection with the biologicalsample, for example any of the MDCK cell lines described above, can beused as the population of cells.

Alternatives to mammalian cell fines as described above for thebiological sample may also be used, e.g., the population of cells of themethod of the invention may be an avian cell line.

In preferred embodiments, the population of cells express anoligonucleotide or polypeptide which is the inhibitory anent of themethod of the invention. Said oligonucleotide may be an oligomericcompound as described in more detail herein below. Preferably, the cellsin said population of cells are transfected and/or engineered to expressthe inhibitory agent. The expression of the inhibitory agent may be,from an expression vector. Vectors and methods for expression ofsequences in cells, in mammalian cells are well known in the art. Theinhibitory anent may be transiently expressed. More preferably, theinhibitory agent is stably expressed in the population of cells. Thatis, a nucleotide sequence, e.g., a DNA sequence, capable of expressingthe inhibitory agent may be transiently transfected into the populationof cells, i.e., the expression of the inhibitory agent in the populationof cells may be by transient transfection of an expression vector.However, more preferably, a nucleotide sequence, a DNA sequence, capableof expressing the inhibitory agent is stably transfected into thepopulation of cells, the expression of the inhibitory agent in thepopulation of cells is by stable transfection, i.e., expression of theinhibitory agent is from a coding sequence stably integrated into thegenome of the cells of said population of cells and/or stably propagatedwithin the population of cells.

If the biological sample contains cells, then the biological sample mayitself serve as the population of cells. In this case, step (i) of theprocesses of the invention will involve contacting the biological samplewith an inhibitory anent, or the biological sample itself may expressthe inhibitory agent, as described above.

Incubation

The invention involves incubating cells (i.e., the population of cellsand/or the biological sample of the process of the invention) underconditions that permit the reproduction of a first intracellularpathogen. Said conditions may also permit the survival, growth,reproduction and/or division of cells within the population of cells.The incubation may be in vivo or in vitro. Specific conditions forreproduction of the first intracellular pathogen and/or permitting cellsurvival, growth, reproduction and/or division of the population ofcells will vary according to normal experiment design.

In preferred embodiments, said conditions may be conditions in which anorganism serves as an incubator for the first intracellular pathogen.

When the population of cells of the invention is a cell culture, theincubating step of the method of the invention preferably involvesincubating (i.e., culturing) the cell culture under conditions thatpermit cell survival, growth, reproduction and/or division. The cellswill be capable, under normal conditions known to the person of ordinaryskill in the area of the invention, of supporting reproduction of one ormore of the intracellular pathogens described above (in the absence ofan inhibitory agent specific to the intracellular pathogen that is to beallowed to reproduce). Thus, the step of further culturing the cellspermits reproduction of pathogens in the culture. Pathogens that arethus allowed to reproduce, or the effects they produce in the culture(e.g. a phenotypic effect upon the population of cells), may then beobserved. According to the methods of the invention, these firstpathogens may be observed preferentially over a second pathogen, whichis also present in the sample, but is inhibited from reproducing by thepresence of a inhibitory agent that is specific for the second pathogen.

In general terms, conditions that permit reproduction of pathogensand/or the growth of suitable cells are well known in the art.Conditions permitting the survival, growth, reproduction and/or divisionof suitable cells are generally provided together with the cells whenobtained from a commercial supplier.

Depending on the cell type and desired assays, cells may be grown insuspension, in adherent culture, or in microcarrier culture. Cells usedwith the invention may thus be adapted for growth in suspension. Onesuitable MDCK cell line that is adapted for growth in suspension cultureis MDCK 33016 (deposited as DSM ACC 2219).

Rather than being grown in the presence of serum, cell lines may begrown in serum-free culture media and/or protein free media. A medium isreferred to as a serum-free medium in the context of the presentinvention in which there are no additives from scrum of human or animalorigin. Protein-free is understood to mean cultures in whichmultiplication of the cells occurs with exclusion of proteins, growthfactors, other protein additives and non-serum proteins, but canoptionally include proteins such as trypsin or other proteases that maybe necessary for supporting replication of certain intracellularpathogens (i.e., the first intracellular pathogen of the methods of theinvention). The cells growing in such cultures naturally containproteins themselves.

In some embodiments, the population of cells may be incubated below 37°C. (e.g. 30-36° C.),

Observation, Testing

According to the invention, material arising from the incubation of thecells under conditions that permit cell growth is tested for firstintracellular pathogen.

Generally, tests may be performed on cells themselves, oncell-containing culture fluid, or on cell-free culture fluid (e.g. on asupernatant of the culture). Such tests include both in vitro and invivo tests. For example, one or more of the following tests may beperformed:

Microscopy, including transmission electron microscopy.

Histological tests, e.g., celluar staining, e.g., histochemical,immunological or immunochemical staining.

A Biochemical tests for retroviruses e.g. a RT assay.

Test for infectivity of a different cell-type e.g. to detect cytopathiceffects.

Molecular genetic tests, such as PCR.

Animal inoculation tests e.g. into adult mice, suckling mice, guineapigs, rabbits.

Infection of embryonated eggs.

The testing may be for a phenotypic effect, e.g., a cytopathic effect,e.g., a plaque in a cell culture, cell death, apoptosis. In somepreferred embodiments, testing may be for inflammation or any phenotypeor biological indicator, mediator or molecule associated withinflammation. Generally, any observable effect may be suitable.

A positive test result, e.g., the presence of a phenotypic or cytopathiceffect, may be indicative of the presence in the biological sample of afirst intracellular pathogen intracellular pathogen other than thesecond intracellular pathogen which cannot reproduce due to the presenceof an inhibitory agent). The absence of an effect that may be attributedto a pathogen is indicative of the absence of the first intracellularpathogen, the absence of an intracellular pathogen that, under theconditions employed, would lead to an observable effect in thepopulation of cells when said pathogen reproduces.

Usually, the invention will be accompanied by one or more controlcultures. E.g. in one form of positive control, the sample and cells arecultured in the absence of the inhibitory agent and/or in the presenceof a known pathogen. In examples of negative controls, the cells may becultured in the absence of one or more pathogens, e.g., the secondintracellular pathogen and/or the first intracellular pathogen, and/orin the absence of the biological sample, or both the sample and theinhibitory agent. Such controls permit ready comparisons to be made.

The Inhibitors Agent

According to the invention, in order to test for a first intracellularpathogen, cells are incubated in the presence of one or more agentinhibiting the survival and/or reproduction of a second intracellularpathogen. If said inhibitory agent(s) were not present, the secondintracellular pathogen would reproduce in the cells, causing phenotypiceffects, and the first and second intracellular pathogens may not beeasily differentiated. The function of the inhibitory agent is toinhibit reproduction of the second intracellular pathogen under theculture conditions, thereby permitting the first intracellular pathogento reproduce in preference to the second intracellular pathogen. Thus,according to the invention, intracellular pathogens may more easily bedifferentiated, and intracellular pathogens of interest the firstintracellular pathogen according to the claims or a further, e.g., athird intracellular pathogen) may more easily be observed.

Preferably, the inhibitory agent does not prevent survival, growth,reproduction and/or division of the population of cells. A slightnegative impact on the cells may be tolerated, but, in the process ofthe invention, cell survival, growth, reproduction and/or division canstill take place under the culture conditions in the presence of theinhibitory agent.

The inhibitory agent also does not prevent survival, multiplicationand/or reproduction of pathogens for which the method is intended totest (e.g., the first intracellular pathogen according to the claims, orone or more further intracellular pathogens which herein may be referredto as third, fourth, fifth or further intracellular pathogens). Again, aslight negative impact may be tolerated, but the process is intended topermit reproduction and detection of the first intracellular pathogen ifit is present in the sample. In the process of the invention, theinhibitory agent inhibits survival and/or reproduction of the secondintracellular pathogen to a greater extent than the agent inhibitssurvival and/or reproduction of the first and/or further intracellularpathogen. That is, the inhibitory agent specifically, selectively orpreferentially inhibits the second intracellular pathogen. Preferably,said inhibitory agent does not, or does not substantially, inhibit thesurvival, multiplication and/or reproduction first (and/or further)intracellular pathogen. Most preferably, the inhibitory agent inhibitssurvival and/or reproduction of the second intracellular pathogencompletely.

When investigating different pathogens, it may thus be possible to usedifferent inhibitory agents. An inhibitory agent may inhibit, orpreferentially, a particular pathogen, but have no effect, or a muchlesser effect, on a, further pathogen, or on further pathogens. In thissituation, the inhibitory agent is suitable for use with the inventionwhere the presence of the first intracellular pathogen of the method ofthe invention is being tested, but because the agent inhibits thereproduction of a second intracellular pathogen, the agent would not besuitable when looking for said second pathogen.

In accordance with the preceding paragraphs, the invention alsocomprises embodiments wherein a second or further agent, inhibiting thereproduction of a third or further intracellular pathogen.

The inhibitory agent may be selected based on the type of intracellularpathogen that is to be inhibited. For example, if the secondintracellular pathogen is a virus, e.g., an influenza virus, theinhibitory agent may be selected based on the type of virus, e.g.,influenza A virus or influenza B virus. In such embodiment, some agentsmay act against both A and B viruses, whereas others are specific.

The inhibitory agent may be an antifungal agent. The inhibitory agentmay also be an antiviral agent or an antibacterial agent. The inhibitoryagent may be an antibiotic.

An inhibitory agent may be selected based on known characteristics ofthe life cycle of the second intracellular pathogen. The secondintracellular pathogen may be lytic pathogen, i.e., it may cause thelysis of cells. When the second intracellular pathogen is lytic, theinhibitory agent preferably inhibits the life cycle of the secondintracellular pathogen such that cell lysis is prevented. For example,in preferred embodiments, the inhibitory agent inhibits replication orgene expression in the second intracellular pathogen. Preferably theinhibitory agent targets one or more RNA species of the secondintracellular pathogen such that reproduction and/or assembly of thesecond intracellular pathogen is prevented. For instance, when thesecond intracellular pathogen is an influenza virus, oseltamivir is lessuseful with the invention than other agents. Oseltamivir is activeagainst influenza virus in vivo, but this activity is exertedextracellularly. As influenza virus is lytic, cell cultures willexperience a large cytopathic effect before the antiviral compound caninterfere with the viral life cycle under the conditions of the process.Generally, inhibitory agents that act extracellularly (includingneuraminidase inhibitors for influenza virus) are not preferred.Preferably, the inhibitory agent inhibits or prevents the intracellularreproduction, growth, multiplication, replication, gene-expression,transcription, and/or translation of the second intracellular pathogen.For example, the inhibitory agent may inhibit gene-expression of thesecond intracellular pathogen. For example, the inhibitory agent maypost-transcriptionally inhibit gene-expression of the secondintracellular pathogen, for example by inhibition of mRNA translation,in particular by degradation of mRNA.

Preferred agents for use with the invention can at least partiallyinhibit or prevent reproduction, growth, multiplication, replication,gene-expression, transcription, and/or translation of an intracellularpathogen. More preferably, said inhibition or prevention is at leastsubstantially complete, most preferably complete.

Preferred agents for use with the invention can at least partiallyinhibit or prevent a phenotypic or empathic effect of the secondintracellular pathogen, e.g., viral cytopathic effects, or can at leastpartially inhibit or prevent hemadsorption and/or hemagglutination. Morepreferably, agents for use with the invention inhibit or prevent viralcytopathic effects, hemadsorption and/or hemagglutination at leastsubstantially completely, most preferably completely.

Suitable inhibitory agents include, but are not limited to, smallorganic compounds (e.g. molecular weight <1000 Da), oligomericcompounds, in particular interfering RNA molecules and antisensemolecules. When the second intracellular pathogen is a virus, suitableinhibitory agents include small molecule antiviral drugs, and peptideinhibitors such as enfuvirtide.

Preferably, the agent comprises an oligomeric compound or moiety, e.g.,a nucleic acid, an oligonucleotide, a nucleic acid derivative or anoligonucleotide derivative, including a modified nucleic acid or amodified oligonucleoside. The agent may inhibit the expression of asequence (e.g., a gene) in the genome of the second intracellularpathogen, that is, the inhibitory agent may be directed against a targetsequence.

Preferably the agent comprises an oligomeric compound or moiety that iscapable of complementary base pairing with the target sequence.Preferably the agent is substantially complementary to a targetsequence. The target sequence may be a nucleotide sequence present in,complementary to, encoded by and/or transcribed from the genome of thesecond intracellular pathogen. Preferably the oligomeric compound iscapable of complementary base pairing with a nucleotide sequencetranscribed from the genome of the second intracellular pathogen, e.g.with an mRNA, e.g. a mature mRNA. If the second intracellular pathogenis a virus, said transcribed sequence may also be within a complementaryRNA or DNA (cRNA or cDNA) genome, i.e., a complementary genome sequencein relation to a plus-strand or minus-strand viral genome. Saidtranscribed sequence may also be a regulatory RNA molecule.

Preferably, the oligomeric compound is 80%, 82%, 84%, 86%, 88%, 90%,92%, 94%, 96%, 98%, 99% or 100% complementary to the target sequence.

The agent comprising an oligomeric compound may be single-stranded ordouble-strand. Likewise, the oligomeric compound may be single-strandedor a double-stranded. When the agent is a double-stranded oligomericcompound—i.e., a duplex—one of the two strands of said agent is capableof said complementary base-pairing with said nucleotide sequence presentin, and/or transcribed from the genome elf the second intracellularpathogen.

A portion or sequence of the agent that is capable of complementarybase-pairing with a target sequence is referred to as being antisense tosaid sequence of said a target sequence. Thus, the agent may be anantisense oligomeric compound. An inhibitory agent that is an antisenseoligomeric compound may act by any antisense-based mechanism of geneinhibition, e.g., an RNAse H, an RNAi (RNA interference) mechanism, aRISC-based mechanism. Herein, a RISC-based mechanism is a mechanisminvolving a RISC complex, including the RNAi mechanism. Agents that actby antisense-based mechanism of gene inhibition are well known in theart, and the person skilled in the art may adapt the structure of theagent accordingly. Preferably, the oligomeric compound acts by RNAi toinhibit the reproduction or the second intracellular agent. Thus, theinhibitory agent may be an interfering &isomeric compound, including aninterfering RNA molecule, such as an siRNA short interfering RNAmolecule).

A strand of an agent that is an oligomeric compound, e.g., anoligonucleotide, may be 19-80 monomers in length, preferably 19-75,19-70, 19-65, 1940, 19-55, 19-50, 19-45, 19-40, 19-35 or 19-30 monomersin length, preferably 19-29, 19-28, 19-27, or 19-26 monomers in length,e.g., 30-26, 21-26, 22-26, 23-26, 19-2520-25, 21-35, 22-25, 23-25,19-24, 20-24, 21-24, 22-24, 23-24, 19-23, 20-23, 21-23, 22-23, 19-22,20-32, 21-22, 19-21, 20-21 or 19-20 nucleotides in length. Said monomersare preferably capable of complementary base pairing with nucleic acids,and preferably comprise nucleic acid bases, nucleosides or nucleotides,e.g., ribonucleosides or deoxyribonucleotides or derivatives thereof.That is, said monomers are preferably capable of complementary basepairing to a target sequence in a manner that supports anantisense-based RNAi) mechanism of inhibiting the expression of thetarget sequence. The agent may be a single strand capable of forming ahairpin structure, that is, by virtue of internal complementary basepairing, e.g., the agent may be a short hairpin RNA.

For example, agents that inhibit coronavirus, e.g., SARD coronavirus orinfluenza virus replication are known in the art. For instance, siRNAmolecules targeting coronovirus are discussed and referenced inreference 24. Reference 25 discloses around 20 different siRNA moleculesthat target influenza A virus. Further siRNA work from the same group isdisclosed in references 26 and 27. Influenza B virus was targeted inreference 28. Further interfering RNAs active against influenza virusare disclosed in references 29 and 30.

Preferably, regions of a virus targeted by oligomeric compounds areconserved among different subtypes and strains of the virus, e.g.,between human, chicken, duck, horse, and/or swine influenza Influenzasequences are available from the influenza sequence database:www.flu.lan1.gov. Preferably, an oligomeric compound does not shareidentity with a known gene in the population of cells used in themethods of the invention, or does not interfere with the ability of thecells to permit the reproduction of said first intracellular pathogen.For example, oligomeric compounds targeting influenza virus may inhibitthe expression of one or more of the HA, NA, M, NP, NS, PA, PB1,PB2,genes. The NP, PA, PB1 and/or PB2 genes are preferred for the selectionof target sequences, in particular the NP and/or the PA genes arepreferred targets.

Antisense nucleic acids active against influenza vitals replication arealso known in the art. For instance, reference 31 discloses antiviralmorpholino antisense oligonucleotides.

Whereas inhibitory agents used in vivo must have acceptable toxicity,pharmacokinetic profiles, half lives, etc., these considerations are notso important with processes of the invention. For instance, there aremany potent antiviral (e.g., anti-influenza) compounds that have beenrejected for routine use in humans because of unfavourable systemicpharmaceutical properties, but which may be used with the invention.Similarly, issues of delivery that are relevant to siRNA or antisensemolecules are less important when dealing with cell cultures. References25 already reports that its siRNA molecules are active against influenzavirus growing in MDCK culture, and reference 28 also focused on culturedcells. Even so, delivery systems can still be used for in vitro work.For instance, delivery of siRNA to cultured cells to inhibit influenzavirus replication was reported in reference 32 using virosomes.Reference 33 further reports the use of polycation-based systems tofacilitate their delivery into cells. Reference 34 used plasmidconstructs to anti-influenza iRNAs in MDCK cultures. Lentiviral vectorswere used with cultured MDCK cells in reference 35.

Preferably, the inhibitory agent is expressed in the population of cellsof the methods of the Invention, e.g., in a cultured cell line or agenetically engineered organism, as described above in connection withthe population of cells. Thus, the inhibitory agent may comprise asingle-stranded and/or double-stranded oligonucleotide that is expressedin the population of cells. As described above, preferably a nucleotidesequence expressing the inhibitory agent is stably propagated in saidpopulation of cells, and the inhibitory agent is stably expressed insaid cell population. In preferred embodiments, the inhibitory agent isexpressed as an oligonucleotide as described above, e.g., as asingle-stranded compound capable of forming a hairpin structure (e.g., ashort hairpin RNA), as two separate strands capable of forming an siRNAduplex, as a single-stranded interfering agent, as an antisense agent,as a compound capable of complementary base pairing with a nucleotidesequence transcribed from the genome of the second intracellularpathogen, as a compound capable of inhibiting reproduction of onintracellular pathogen by an antisense, e.g., an RNA i mechanism, etc.Preferably, the inhibitory agent is a hairpin RNA. Thus, the populationof cells preferably contains a nucleotide sequence encoding andexpressing the inhibitory agent of the methods of the invention. Theinhibitory agent is preferably a hairpin RNA and/or comprises anantisense RNA.

For example, siRNA expression vectors have been described in reference34.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantial” does not exclude “completely” e.g. a compositionwhich is “substantially free” from Y may be completely free from Y.Where necessary, the word “substantially” may be omitted from thedefinition of the invention.

The term “about” in relation to a numerical value x means, for example,x±10%.

The term “one or more” encompasses “one”, “more than one”, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, . . . etc.

The term “two or more” encompasses “two”, “more than two”, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, . . . etc.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined, with the third component, etc.

BRIEF DESCRIPTION OF DRAWINGS

There are no drawings.

MODES FOR CARRYING OUT THE INVENTION Example

In one preferred embodiment of the method of the invention, a biologicalsample derived from a MDCK cell culture contains influenza virus. Thesample is contacted with a uninfected (pathogen. free) population ofMDCK cells (a test culture) in a plaque assay. Reproduction of theinfluenza virus in the test culture is inhibited by RNAi, which allowsplaque formation in the test culture to be observed and attributed to apathogen other than the influenza virus.

The test culture may be a monolayer of MDCK cells in 1% semisolid agar.An appropriate amount of siRNA directed against a suitable influenzavirus target sequence, e.g., the influenza virus nucleoprotein (NP) oracidic polymerase (PA gene, is introduced into the cells of the testculture (e.g. 2.5 nmol per 1×10⁷ MDCK cells) by methods known in theart. The siRNA sequences may be, for example, as disclosed in references25 and 27, i.e., sense oligonucleotide 5′-GGAUCUUAUUUCUUCGGAGdTdT-3′(SEQ ID NO: 1) and antisense oligonucleotide5-dTdTCCUAGAAUAAAGAAGCCUC-3′ (SEQ ID NO; 2) directed against thenucleoprotein (NP) gene; or sense oligonucleotide5′-GCAAUUGAGGAGUGCCUCAdTdT-3′ (SEQ ID NO: 3), and antisenseoligonucleotide 5′-dTdTCGUUAACUCCUCACGGACU-3 (SEQ ID NO: 4) directedagainst the acidic polymerase gene. After 7-10 hours, appropriateamounts of the sample (e.g., 2-fold or 10-fold serial dilutions) areadded to test cultures. The titer or content of influenza virus in thetest culture and/or the inhibition of influenza inhibition by the siRNAmay be monitored, for example, by subjecting serial dilutions of testculture supernatant to a haemagglutation (HA) assay, or by RNAextraction, reverse transcription and PCR employing methods that arewell known in the art. After a suitable incubation time, e.g, two days,the test culture is assessed for plaque formation by staining withcrystal violet. If the influenza virus does not reproduce duringincubation, any plaques visualized by the staining are attributable to apathogen other than the influenza virus.

In addition to the controls mentioned above, siRNA specific for GFP mayalso be introduced into GFP-expressing MDCK cells followed by virusinfection (e.g., with influenza virus) or addition of the tested sample.GFP expression may be assayed by flow cytometry.

Materials and Methods

MDCK cells are grown by methods well known in the art, e.g., in OWNcontaining 10% heat-inactivated FCS, 2 mM 1-glutamine, 100 units/mlpenicillin, and 100 μg/ml streptomycin at 37° C. under a 5% CO2/95% airatmosphere.

Influenza virus stocks are grown in the allantoic cavity of 10-day-oldembryonated chicken eggs (Charles River Laboratories, Wilmington, Mass.)at 37C. Allantoic fluid is harvested 48 h after virus inoculation andstored at −80° C. Virus titer is measured by hemagglutination or plaqueassays.

RNA oligonucleotides for siRNAs are commercially available or may bechemically synthesized according to methods known in the art. In orderto obtain a duplex siRNA, equimolar amounts of complementaryoligonucleotides are mixed, heated to 95° C. for 5 min, then annealed byreducing the temperature, e.g., by 1° C. every 30 sec until 35° C., thenby 1° C. every min until 5° C. siRNA duplexes are analyzed forcompletion of duplex formation by gel electrophoresis.

The suitability and efficacy of siRNAs inhibiting the reproduction ofthe second pathogen, e.g., influenza virus, may be assessed separatelyin MDCK cells. Logarithmic-phase MDCK cells may be trypsinized, washed,and resuspended in serum-free medium, e.g., RPMI medium 1640, at 2×10⁷cells per ml. Cells (0.5 ml) are mixed with siRNA and electroporated at400 V and 975 μF by using a Gene Pulser apparatus (Bio-Rad).Electroporated cells are divided and cultured in DMEM for 8 h. Theculture medium is then removed and a suitable amount of virus in DMEM,0.3% BSA (Sigma), 10 mM Hepes, 100 units/ml penicillin, and 100 μg/mlstreptomycin, is added to each well. After incubation for 1 h at roomtemperature, 2 ml of DMEM buffer containing 0.3% BSA (Sigma), 10 mMHepes, 100 units/ml penicillin, 100 streptomycin, and 4 μg/ml trypsin isadded to each Cells are then cultured at 37° C. under 5% CO and thevirus titer monitored in the supernatant.

The hemagglutination assay is carried out in V-bottom 96-well plates,Serial dilutions (e.g., 2-fold) of samples are mixed with an equalvolume of a 0.5% suspension (vol/vol) of erythrocytes, e.g., chickenerythrocytes available from Charles River Laboratories). Afterincubation on ice for one hour, wells are assessed for adherent,homogeneous layers of erythrocytes, which indicate hemagglutination.

For plaque assays, serial dilutions of samples are added onto monolayersof MDCK cells in 1% semisolid agar. After two days, plaques arevisualized by staining with crystal violet.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES

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[13] Pau et al. (2001) Vaccine 19:2716-21.

[14] http://www.atcc.org/

[15] http://locus.umdnj.edu/

[16] EP-A-1260581 (WO01/64846).

[17] WO2006/071563.

[18] WO20051113758.

[19] WO03/076601.

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[35] Hui et (2004) J Gen Virol. 85; 1877-84.

1-20. (canceled)
 21. A process for testing for a first intracellularpathogen in a biological sample, comprising the steps of: (i) contactingthe sample with a population of cells in the presence of an inhibitoryagent, wherein the inhibitory agent specifically inhibits thereproduction of a second intracellular pathogen that is known to bepresent in the sample; (ii) incubating the cells under conditions thatpermit the reproduction of the first intracellular pathogen while thesecond intracellular pathogen is inhibited from reproducing by thepresence of the inhibitory agent; and (iii) testing material arisingfrom step (ii) for the first intracellular pathogen by transmissionelectron microscopy, a reverse transcription-based biochemical test forretroviruses, detection of a cytopathic effect, a PCR-based moleculargenetic test, an animal inoculation test or infection of embryonatedeggs.
 22. The process of claim 21, wherein the first intracellularpathogen comprises a prokaryote, a eukaryote and/or a virus.
 23. Theprocess of claim 21, wherein the second intracellular pathogen comprisesa prokaryote, a eukaryote and/or a virus.
 24. The process of claim 21,wherein the second intracellular pathogen comprises a virus.
 25. Theprocess of claim 21, wherein the first intracellular pathogen comprisesone or more pathogens selected from Bartonella, Bordetella, Brucella,Chlamydiae, Chlamydia, Chlamydophila, Ehrlichia, Escherichia,Francisella, Legionella, Listeria, Mycobacterium, Mycobacterium,Rickettsiae, Salmonella, Shigella, Streptococcus, Yersinia, Babesia,Cryptococcus, Cryptosporidium, Eimeria, Histoplasma, Leishmania,Plasmodium, Theileria, Toxoplasma, Trypanosoma, Pneumovirinae, thePneumovirus genus, respiratory syncytial virus (RSV), Morbilliviruses ofthe Paramyxoviridae family, the measles virus, Enteroviruses of thePicornaviridae family, Coxsackie viruses, echoviruses and enteroviruses,mammalian Reoviridae, avian Reoviridae, orthoreoviruses, rotaviruses,Metapneumoviruses of the Paramyxoviridae family, human metapneumovirus(HMPV), Rubulaviruses of the Paramyxoviridae family, mumps virus,Togaviridae, Rubella virus, Coronaviridae, human coronaviruses, SARScoronavirus, Rhinoviruses of the Picornaviridae family, M-strains ofRhinovirus, Varicella Zoster virus (VZV), human herpes virus 2 (HHV3),Polyomaviridae, SV-40 polyomavirus, BK polyomavirus, JC polyomavirus,porcine circoviruses, porcine picornaviruses, swine vesicular diseasevirus (SVDV), Teschen-Talfan virus, Parvoviruses, canine parvovirus(CPV), porcine parvoviruses, Bocaviruses, Orthomyxoviridae, influenza Avirus, influenza B virus, influenza C virus, Parainfluenza viruses(PIV), Paramyxoviridae paramyvovirinae, Parainfluenza virus type 1,Parainfluenza virus type 2, Parainfluenza virus type 3, Parainfluenzavirus type 4, Parainfluenza virus type 5, Herpesviridae, herpes simplexvirus 1 and 2, Adenoviridae, adenoviruses, human and simian adenovirus,avian circoviruses, bimaviridae, and infectious bursal disease virus(also known as gumboro virus).
 26. The process of claim 21, wherein saidsecond intracellular pathogen comprises a pathogen selected fromBartonella, Bordetella, Brucella, Chlamydiae, Chlamydia, Chlamydophila,Ehrlichia, Escherichia, Francisella, Legionella, Listeria,Mycobacterium, Mycobacterium, Rickettsiae, Salmonella, Shigella,Streptococcus, Yersinia, Babesia, Cryptococcus, Cryptosporidium,Eimeria, Histoplasma, Leishmania, Plasmodium, Theileria, Toxoplasma,Trypanosoma, Pneumovirinae, the Pneumovirus genus, respiratory syncytialvirus (RSV), Morbilliviruses of the Paramyxoviridae family, the measlesvirus, Enteroviruses of the Picornaviridae family, Coxsackie viruses,echoviruses and enteroviruses, mammalian Reoviridae, avian Reoviridae,orthoreoviruses, rotaviruses, Metapneumoviruses of the Paramyxoviridaefamily, human metapneumovirus (HMPV), Rubulaviruses of theParamyxoviridae family, mumps virus, Togaviridae, Rubella virus,Coronaviridae, human coronaviruses, SARS coronavirus, Rhinoviruses ofthe Picornaviridae family, M-strains of Rhinovirus, Varicella Zostervirus (VZV), human herpes virus 2 (HHV3), Polyomaviridae, SV-40polyomavirus, BK polyomavirus, JC polyomavirus, porcine circoviruses,porcine picornaviruses, swine vesicular disease virus (SVDV),Teschen-Talfan virus, Parvoviruses, canine parvovirus (CPV), porcineparvoviruses, Bocaviruses, Orthomyxoviridae, influenza A virus,influenza B virus, influenza C virus, Parainfluenza viruses (PIV),Paramyxoviridae paramyvovirinae, Parainfluenza virus type 1,Parainfluenza virus type 2, Parainfluenza virus type 3, Parainfluenzavirus type 4, Parainfluenza virus type 5, Herpesviridae, herpes simplexvirus 1 and 2, Adenoviridae, adenoviruses, human and simian adenovirus,avian circoviruses, bimaviridae, and infectious bursal disease virus(also known as gumboro virus).
 27. The process of claim 21, wherein itis unknown whether the first intracellular pathogen is present in thebiological sample to be tested.
 28. The process of claim 21, wherein thesecond intracellular pathogen is a virus.
 29. The process of claim 21,wherein one or both of the biological sample and the population of cellscomprise or are derived from mammalian cells.
 30. The process of claim29, wherein the mammalian cells comprise one or more of hamster, cattle,primate, human, monkey and dog cells.
 31. The process of claim 29,wherein the mammalian cells comprise one or more of kidney cells,fibroblasts, retinal cells, liver cells, lung cells.
 32. The process ofclaim 29, wherein the mammalian cells are MDCK cells.
 33. The process ofclaim 21, wherein the inhibitory agent comprises an oligomeric compound.34. The process of claim 33, wherein the oligomeric compound is capableof complementary base pairing to a nucleotide sequence present in,encoded by and/or transcribed from the genome of the secondintracellular pathogen.
 35. The process of claim 34, wherein theoligomeric compound acts by RNAi to inhibit the reproduction of thesecond intracellular pathogen.
 36. The process of claim 35, wherein theinhibitory agent comprising the oligomeric compound is double-stranded.37. The process of claim 21, wherein the inhibitory agent is anantifungal agent.
 38. The process of claim 21, wherein the inhibitoryagent is an antiviral agent.
 39. The process of claim 21, wherein theinhibitory agent is an antibacterial agent.
 40. The process of claim 21,wherein a test result of step (iii) is obtained within 12 months. 41.The process of claim 21, wherein the inhibitory agent specificallyinhibits the intracellular reproduction of the second intracellularpathogen.